The eukaryotic ATP-ases of SMC family (structural maintenance of chromosomes) form several essential protein complexes that determine the higher-order chromosome structure and dynamics in eukaryotic cells. One of these complexes, termed condensin, is in the current focus of studies by the Unit. Condensin complex constitutes the main molecular machine of chromosome condensation, a process indispensable for proper segregation of sister chromatids during cell division. Condensin is conserved in eukaryotic evolution, and in budding yeast it is composed of five essential subunits: Smc2, Smc4, Ycs5/Ycg1, Ycs4 and Brn1. The vital activity of condensin is likely to bind DNA and to change its superhelical state, introducing positive DNA supercoiling. Existing in vitro studies do not allow, however, to predict how condensin interacts with a chromatin fiber - its natural substrate. Thus, the characterization of the authentic binding sites for condensin activity in vivo is an essential step towards elucidating the molecular mechanisms of chromosome condensation. In order to understand the essence of condensin activity in chromatin the studies in the Unit were focused on several particular readouts of this activity, more recently on (1) the specificity of condensin targeting to the nucleolar chromatin and (2) the role of active condensin for functionality of other chromosomal sites (recently identified in the Unit using a whole-genome analysis), specifically at centromeres.[unreadable] (1) The previous characterization by this Unit of the S. cerevisiae rDNA as a main target of mitotic condensin activity could have identified one of the key applications of condensin activity in all eukaryotes. In order to answer the question about the nature of the special role played by condensin in the nucleolus, first, it was tested whether condensin is required to segregate a single rDNA repeat placed on a circular minichromosome. The analysis of such a minichromosome has shown that condensin inactivation results in up to a 20% decrease in its mitotic stability, as compared to either rDNA-less minichromosome in the same mutant or to the rDNA minichromosome in wild type. This finding establishes that even a single rDNA repeat requires condensin activity for proper segregation. It also offers a plausible explanation for the hypersensitivity of the tandem rDNA locus for condensin proficiency: a cumulative effect of multiple individual repeats, each having the abovementioned decrease in segregation fidelity.[unreadable] In order to find the molecular determinants of condensin requirement in rDNA, condensin binding sites in rDNA were analyzed by quantitative ChIP and two of them were found positioned within the Pol I-transcribed rRNA gene. As the Pol I transcription is very active, its physical cohabitation with condensin binding could be problematic. To solve this problem, condensin occupancy was monitored in parallel with 35S transcript levels in a time-course experiment. All four condensin peaks showed similar cell cycle profiles of DNA association, but the intensity of condensin binding was inversely correlated with the level of Pol I transcript at a given timepoint. This counter-dependence of condensin binding and the transcription level in rDNA was confirmed in direct experiments modulating transcription of rDNA by both Pol I (rapamycin treatment) and Pol II (GAL promoter-controlled rDNA). Thus, condensin binding and transcription in rDNA are mutually exclusive. Therefore, condensin must directly compete for binding sites with transcription machinery in rDNA.[unreadable] The competition between condensin and transcription in the native rDNA locus can, however, be resolved by the compartmentalization of transcriptionally silent units and transcriptionally active repeats, which is indeed the case in budding yeast. A compelling evidence was found in favor of this compartmentalization hypothesis, by modulating transcription levels in rDNA with simultaneous monitoring of condensin occupancy and/or rDNA segregation fidelity. Indeed, Pol I transcription and segregation proficiency of rDNA are inversely correlated, with segregation being largely dependent on condensin function. In the independent time-lapse microscopy experiment it was shown that the segregation of chromosome XII with the minimal-size (20-repeat) rDNA array, where all repeats are actively transcribed, is significantly delayed compared to the wild type. Thus, the probable mechanism behind special condensin requirements in rDNA segregation in S. cerevisiae is the need to segregate the actively transcribed and fully assembled nucleolus, probably by counteracting the DNA over-winding in the rDNA regions flanking the actively transcribed repeats. This study revealed a likely functional role for previously unexplained Pol I transcription heterogeneity of native rDNA repeats. [unreadable] (2) The ChIP-chip analysis of the genome-wide condensin binding pattern, previously conducted in the Unit, showed that the peri-centromeric regions are enriched in condensin binding. This enrichment suggests that condensins activity may promote centromere function. Other experimental facts also hint that condensin has some role at centromeres. For example, in budding yeast, condensin mutants maintain high viability throughout the mitotic arrest, indicative of a checkpoint control, possibly mediated by kinetochores. Upon biochemical and cytological analysis, it was shown that the spindle assembly checkpoint (SAC) is indeed the factor responsible for condensin mutant arrest. The dependence of condensin mutant arrest on SAC was not allele-specific, indicating that condensin function as a whole is monitored by SAC. Moreover, the depletion of condensin in human cells engages the MAD2-dependent spindle checkpoint as well, resulting in a transient metaphase delay.[unreadable] This discovery provided an opportunity to elucidate the mitotic condensin function, which is not obscured by SAC. In particular, it was interesting to explain the low mitotic stability of the rDNA-containing chromosomes in condensin mutants. After comparing individual rates of missegregation for the rDNA-containing chromosome XII and two non-rDNA chromosomes, by employing in vivo chromosomal fluorescent tags, it shown that in the smc2-8 mutant the rDNA-containing chromosome XII was much more prone to missegregation than chromosomes IV or IX in the SAC-proficient cells. However, the equally high missegregation rates were observed for all three chromosomes in the SAC-deficient double mutant smc2-8 bub1&#8710;. Thus, condensin is essential for the segregation of the whole yeast genome. Even more compelling evidence that condensin triggers SAC was obtained by showing that double condensin mutants with sgo1&#8710; and skp1-AA alleles, both impairing the sensor of improper inter-centromere tension, result in lethality. Moreover, a direct measurement of inter-kinetochore distance in metaphase showed that it is markedly increased in condensin mutants, compared to metaphase in wild type.[unreadable] The possible nature of the initial SAC trigger in the smc2 mutant was examined by analysis of centromere and kinetochore composition. It showed that the Dsn1 protein (a part of the MIND complex) and the Cse4 protein (the orthologue of the CENP-A centromeric histone) were partially delocalized from centromeres; whereas DDD, COMA, Ndc80 complexes and inner kinetochore proteins were not visibly affected. Thus, centromeric chromatin is the likely mediator between condensin activity and the kinetochore. Therefore, it is plausible that condensins contributes to the proper tension between sister kinetochores by establishing localized condensation of the centromeric region, which is enclosed between the two closest cohesion sites. This analysis has uncovered a putative molecular interface between condensin and centromeres.